Sialyltransferase and DNA encoding the same

ABSTRACT

A sialyltransferase having the following physico-chemical properties;  
     (1) Activity:  
     transfers sialic acid from a sialic acid donor selectively to a 3-hydroxyl group of a galactose residue contained in lactosylceramide as a sialic acid acceptor to produce ganglioside G M3 ;  
     (2) Optimal reaction pH:  
     6.0 to 7.0; and  
     (3) Inhibition and activation:  
     the activity increases at least 1.5 times with 10 mm of Mn 2+  as compared with the case in the absence thereof.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a continuation-in-part of U.S. Ser No. 09/112,563 filedJul. 9, 1998.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to a sialyltransferase and to a DNAencoding the same. More particularly, the present invention relates toan enzyme which synthesizes ganglioside G_(M3) by transferring sialicacid to a galactose residue of lactosylceramide and to a DNA encodingthe enzyme.

[0003] Human myelogenous leukemia cell line HL-60, which is a cell linethat has acquired the ability of infinite proliferation as a result oftumorigenic transformation, is used generally and widely as a model forleukemia cells (Collins, S. J. Gallo, R. C., and Gallagher, R. E.,Nature (London), 270, 347-349 (1977); Collins, S. J., Blood, 70, 1223(1987)). The cell line does not differentiate even after continuedcultivation and continues to proliferate while it remains asundifferentiated cells. However, when cultivation is continued withaddition of phorbol ester, which is widely used as a differentiationinducer, the cell line stops the proliferation of cells and takes anappearance similar to that of monocytes or macrophages. This indicatesthat differentiation has been induced. It has been reported that duringthis process, the amount of G_(M3), which is a kind of ganglioside,increases considerably (Nojiri, E., Takaku, F., Tetsuka, T., and Saito,M., Blood, 64, 534-541(1984)), and when the ganglioside G_(M3) is addedexogenously, the cell line shows the same change as that observed withthe addition of phorbol ester, i.e., the cells undergo monocyticdifferentiation (Saito, M., Terui, Y., and Nojiri, H., Biochem Biophys.Res. Commun., 132, 223-231 (1985)). Also, it has been proved that inthis differentiation process, G_(M3) itself has an activity of inducingdifferentiation (Nojiri, H., Takaku, F., Miura, Y., and Saito, M., Proc.Natl. Acad. Sci. U.S.A., 83, 782-786 (1986)), and that chemicallysynthesized G_(M3) also induces differentiation (Sugimoto, M. and Ogawa,T., Glycoconi. J., 2, 5-9 (1985); Saito, M., Nojiri, H., Ogino, H., Yuo,A., Ogura, H., Itoh, M., Tomita, K., Ogawa, T., Nagai, Y., and Kitagawa,S., FEBS Lett., 271, 85-88 (1990)).

[0004] On the other hand, it has been elucidated that sialicacid-containing glycolipids, in particular ganglioside, bear importantfunctions in various biological phenomena and not only its functions butalso its biosynthesis are being clarified. In vertebrates, manygangliosides (ganglio-series gangliosides) have a common precursor,G_(M3), which has the simplest structure among major gangliosides andthe G_(M3) synthesis affords a basis for the biosynthesis ofgangliosides which have major functions.

[0005] As described above, ganglioside G_(M3) itself participates in theproliferation/differentiation of cells and tissues and it is suggestedthat the ganglioside G_(M3) is a precursor for a group of highergangliosides having various functions in vertebrates.

[0006] G_(M3) has been considered to be synthesized fromlactosylceramide by transfer of sialic acid to the galactose residue inlactosylceramide by CMP-sialic acid:lactosylceramide sialyltransferase(CMP-NeuAc: Galβ1-4Glcβ1-1′Cerα2,3-sialyltransferase; SAT-1). However,neither the transferase from mouse and human has been isolated nor thegenes thereof have been identified.

[0007] Enzymes which transfer sialic acid through an a2-3 ketoside bondare described in, for example, Wienstein et al., J. Biol. Chem., 257,13835 (1982); Gillespie et al., Glycoconj., 7, 469 (1990); Gillespie,W., Kelm, S. and Paulson, J C., J. Biol. Chem., 267, p21001-21010(1992); Lee, Y C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T.,Hashimoto, T. and Tsuji, S., J. Biol. Chem., 269, p10028-10033 (1994);Kim. Y J., Kim, K S., Kim, S H., Kim, C H., Ko, J H., Choe, I S., Tsuji,S. and Lee, Y C., Biochem. Biophys. Res. Commun., 228, p324-327 (1996);and JP-A 5-336963. However, none of the enzymes is known to be involvedin the synthesis of G_(M3) or shows an enzyme activity of transferringsialic acid to lactosylceramide through an α2-3 ketoside bond. Sandhoff,K. et al. presume that α2-3 sialyltransferase (SAT4) is identical withthe enzyme which synthesizes G_(M3) (J. Biol. Chem., 268, 5341 (1993)).However, this is a presumption based on an indirect method, which failsto support that the enzymes are identical to each other as a substance.

[0008] In spite of various attempts which have been made in order toelucidate and control its biosynthesis according as the clarification ofimportance of ganglioside G_(M3) proceeds, the above-mentionedsialyltransferase, which relates closely to the synthesis of G_(M3), hasnot been isolated yet from mouse and human because of difficulty inpreparing the enzyme protein and, hence, neither its gene expressioncontrol mechanism has been clarified yet nor its proteo-chemical orenzymological analysis has been performed successfully.

SUMMARY OF THE INVENTION

[0009] As a result of intensive investigation with view to elucidatingthe control mechanism of cell differentiation by carrying forwardstudies on gene expression control mechanism of and proteo-chemical andenzymological analyses of the above-mentioned sialyltransferase, thepresent inventors have been successful in isolating cDNA having anucleotide sequence encoding the sialyltransferase which participates inthe above-mentioned G_(M3) synthesis from mouse and human, by using anexpression cloning method and based on the nucleotide sequence, theyhave clarified the structure of the above-mentioned sialyltransferase.As a result, it revealed that the enzyme is low in homology with theknown sialyltransferase and is believed to be a new enzyme, differingfrom the α2-8 sialyltransferase, with which the identity was presumed bySandhoff, K. supra.

[0010] Accordingly, the present invention provides a sialyltransferasehaving the following properties and a DNA having a nucleotide sequenceencoding it.

[0011] (1) Activity:

[0012] The sialyltransferase transfers sialic acid from a sialic aciddonor selectively to a 3-hydroxyl group of a galactose residue containedin lactosylceramide as a sialic acid acceptor to produce gangliosideG_(M3).

[0013] (2) Optimal reaction pH:

[0014] 6.0 to 7.0.

[0015] (3) Activation:

[0016] The activity increases at least 1.5 times with 10 mM of Mn²⁺ ascompared with the case in the absence thereof.

[0017] Also, the present invention provides a sialyltransferase havingthe above-mentioned activity and having a C-terminal amino acid sequenceshown by SEQ ID NO: 5 and a DNA encoding it as well as asialyltransferase having the above-mentioned activity and having anamino acid sequence shown by SEQ ID NO: 6 or 11 and/or 12 and a DNAencoding it.

[0018] The sialic acid donor is preferably cytidine5-monophosphsate-sialic acid (CMP-sialic acid).

[0019] The above-mentioned enzymes and DNAs are preferably those derivedfrom a mammal, most preferably those derived from human.

[0020] The present invention also provides a sialyltransferasecomprising the polypeptide (a) or the polypeptide (b) below and a DNAencoding it.

[0021] (a) A polypeptide having an amino sequence shown by SEQ ID NO: 2or 8.

[0022] (b) A polypeptide having an amino acid sequence (a) above, whichhas therein substitution, deletion, insertion or rearrangement of one ora few amino acid residues, said sialyltransferase having an enzymeactivity of transferring sialic acid from a sialic acid donorselectively to the 3-hydroxyl group of galactose residue contained inlactosylceramide as a sialic acid acceptor to produce gangliosideG_(M3).

[0023] Specific examples of the DNA of the present invention include aDNA having a nucleotide sequence encoding all the amino acid sequenceshown by SEQ ID NO: 2 or 8, or a DNA having partial sequences thereof,for example, DNA having a nucleotide sequence shown by SEQ ID NO: 1 or7.

[0024] Further, the present invention provides a polypeptide comprisingall or part of the polypeptide of sialyltransferase encoded by thenucleotide sequence of the above-mentioned DNA From the polypeptide, atransmembrane domain may be deleted.

[0025] In addition, the present invention provides a recombinant vectorcomprising the DNA of the present invention; a transformant into whichthe DNA of the present invention is introduced, and in which the DNA canbe expressed; and a method for producing a sialyltransferase or apolypeptide thereof, comprising cultivating the transformant as definedabove in a suitable medium, to produce and accumulate in the culture thesialyltransferase or the polypeptide thereof encoded by the DNA, andcollecting the sialyltransferase or the polypeptide thereof from theculture.

[0026] The phrase “encoding an enzyme” as used herein refers to encodingthe polypeptide of the enzyme. Also, herein, the sialyltransferase ofthe present invention which has an enzyme activity of transferringsialic acid from a sialic acid donor selectively to the 3-hydroxyl groupof the galactose residue contained in lactosylceramide as a sialic acidacceptor to form α2-3 linkage, thereby producing ganglioside G_(M3), isalso described as “sialyltransferase-1” or “SAT-1” for convenience'ssake.

[0027] According to the present invention, a DNA of α2-3sialyltransferase (SAT-1) which synthesize from lactosylceramide,ganglioside G_(M3) that induces cell differentiation. According to thepresent invention, α2-3 sialyltransferase, i.e., G_(M3) synthase, can beobtained easily by the use of the above-mentioned DNA.

[0028] Since the DNA encoding SAT-1 is provided by the presentinvention, the elucidation of expression mechanism thereof will give anexpectation for elucidation of the mechanism of cell differentiation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a schematic view showing the structure of mouse α2-3sialyltransferase (SAT-1) of the present invention, in which Δ indicatesan N-glycosylation site presumed from the amino acid sequence and TMindicates a transmembrane domain presumed from the amino acid sequence.

[0030]FIG. 2 is a diagram comparing the amino acid sequence of asialylmotif (L and S) region of mouse SAT-1 with a sialylmotif region ofanother sialyltransferase; the marks “*” under the sequences indicatecommon sequences appearing in the sialylmotif of othersialyltransferases, the marks “*” above the sequences indicate the partof mouse SAT-1 that contains the amino acids identical with the aminoacids in the common sequences of the sialylmotif, and the marks “−”above the sequences indicate the part that contains amino acidsdifferent from those in the common sequences of the sialylmotif.

[0031]FIG. 3 is a schematic view showing the structure of human α2-3sialyltransferase (SAT-1) of the present invention, in which Δ indicatesan N-glycosylation site presumed from the amino acid sequence and TMindicates a transmembrane domain presumed from the amino acid sequence.

[0032]FIG. 4 is a diagram comparing the amino acid sequence of asialylmotif (L and S) region of human SAT-1 with a sialylmotif region ofanother sialyltransferase.

[0033]FIG. 5 is a graph illustrating hydropathy-plot of an amino acidsequence of human SAT-1 deduced from a nucleotide sequence of the DNA ofthe present invention.

[0034]FIG. 6 is a graph showing results of flow analyses of gangliosideG_(M3) expression in mouse lung carcinoma cells.

DETAILED DESCRIPTION OF THE INVENTION

[0035] [1] Sialyltransferase-1 of the Present Invention (Enzyme of thePresent Invention) and DNA Encoding the Same (DNA of the PresentInvention)

[0036] The enzyme of the present invention includes sialyltransferaseshaving the following activity:

[0037] (1) Activity;

[0038] The sialyltransferase selectively transfers sialic acid from asialic acid donor to a 3-hydroxyl group of a galactose residue containedin lactosylceramide as a sialic acid acceptor, to produce gangliosideG_(M3). In other words, the enzyme does not transfer substantiallysialic acid to a position except for a 3-hydroxyl group of a galactoseresidue of the above-mentioned sialic acid acceptor. The sialic acidacceptor is preferably CMP-sialic acid.

[0039] Preferably, the enzyme of the present invention further has thefollowing physico-chemical properties:

[0040] (2) Optimal Reaction pH:

[0041] This enzyme has high sialic acid transferring activity within therange of an enzyme reaction mixture pH of from 6.0 to 7.0 as measured bythe enzyme activity assay method described in the examples below.

[0042] (3) Activation:

[0043] The activity of sialyltransferase increases at least 1.5 times inthe presence of 10 mM of Mn²⁺ as compared with the case in the absencethereof.

[0044] Further, the enzyme of the present invention includessialyltransferases having an activity of transferring sialic acid to a3-hydroxyl group of a galactose residue (preferably, the activity (1)above) and having a C-terminal amino acid sequence shown by SEQ ID NO: 5as well as sialyltransferases having an activity of transferring sialicacid to a 3-hydroxyl group of a galactose residue (preferably, theactivity (1) above) and having an amino acid sequence shown by SEQ IDNO: 6 or 11 and/or 12. The amino acid sequence shown by SEQ ID NO: 6 or11 is a sequence which corresponds to a sialylmotif (sialylmotif L)existing in the sialyltransferase and which usually exists in the partcorresponding to the amino acid numbers 136-183 in the amino acidsequence shown by SEQ ID NO: 2 or 8 in the amino acid sequence of thepolypeptide of the sialyltransferase. The amino acid sequence shown bySEQ ID NO: 12 is a sequence which corresponds to another sialylmotif(sialylmotif S) existing in the sialyltransferase and which usuallyexists in the part corresponding to the amino acid numbers 283-305 inthe amino acid sequence shown by SEQ ID NO: 2 or 8 in the amino acidsequence of the polypeptide of the sialyltransferase.

[0045] Specific examples of the polypeptide of the enzyme of the presentinvention includes those of amino acid numbers 38-359 or 1-359 in theamino acid sequence shown by SEQ ID NO: 2 and amino acid numbers 41-362or 1-362 in the amino acid sequence shown by SEQ ID NO: 8.

[0046] The DNA of the present invention is not limited particularly asfar as they encode those polypeptides and includes those encoding thepolypeptides (a) or (b) below.

[0047] (a) A polypeptide having an amino sequence shown by SEQ ID NO: 2or 8.

[0048] (b) A polypeptides having the amino acid sequence (a) above,which has therein substitution, deletion, insertion or rearrangement ofone or a few amino acid residues and having an enzyme activity oftransferring sialic acid to a 3-hydroxyl group of a galactose residue(preferably the activity (1) above).

[0049] In other words, the amino acid sequence shown by SEQ ID NO; 2 mayhave therein substitution, deletion, insertion or rearrangement of oneor a few amino acid residues that do not substantially impair theactivity of transferring sialic acid to a 3-hydroxyl group of agalactose residue (preferably the activity (1) above). The DNA of thepresent invention includes DNAs having any of substitution, deletion,insertion and rearrangement in their nucleotide sequence, encoding suchpolypeptides having therein any of substitution, deletion, insertion andrearrangement in their amino acid sequence. The term “a few amino acidresidues” as used herein refers to the number of amino acids that maycause variations to the extent that the activity of the enzyme is notlost. In the case of a polypeptide consisting of 360 amino acidresidues, for example, it means about 20 or less, preferably about 10 orless. The activity of the enzyme can be measured without difficulty by aknown method (JP-A 7-327678) by changing cDNA to be introduced into hostcells and a substrate for the enzyme and since one skilled in the artcan practice with ease, for example, by the method specificallydescribed herein, the substitution, deletion, insertion or rearrangementof one or a few amino acid residues that does not substantially impartthe target activity can be readily selected by using the presence orabsence of the enzyme activity as an indicator. The substitution,deletion, insertion or rearrangement in the nucleotide sequence can beintroduced into a DNA by synthesizing a sequence having a restrictionenzyme-cleaved end on each terminal and containing both sides of themutation point, i.e., substitution, deletion, insertion orrearrangement, followed by replacing this for the corresponding part ofthe nucleotide sequence of a non-mutated DNA Alternatively,site-specific mutagenesis (Kramer, W. and Frits, H. J., Meth. inEnzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154,367 (1987)) and the like can be used to introduce substitution,deletion, insertion or rearrangement into a DNA. Also, the DNA encodinga polypeptide having substitution, deletion, insertion or rearrangementof one or a few amino acid residues that does not substantially impairthe activity in the amino acid sequence shown by SEQ ID NO: 2, may beobtained as a homologous or allelic variant.

[0050] The amino acid sequences shown by SEQ ID NO: 2 and SEQ ID NO: 8are derived from mouse and human, respectively, it is predicted thatthere is a difference which does not affect the activity betweenindividuals of each species. The substitution, deletion, insertion orrearrangement one or a few amino acid that does not substantially impairthe activity is preferably within a range of mutation betweenindividuals.

[0051] Specifically, the DNA of the present invention includes DNAshaving nucleotide sequences encoding all the amino acid sequence shownby SEQ ID NO: 2 or 8, or DNAs having partial nucleotide sequencesthereof. These DNAs are preferred but the present invention is notlimited thereto. The term “DNAs having partial nucleotide sequences” asused herein refers to, for example, those DNAs which hybridize with DNAencoding a polypeptide of mouse or human sialyltransferase-1 (inparticular, a part of amino acid numbers 30-362, 38-362, 41-362 or136-183 in the amino acid sequence of SEQ ID NO: 8) so that they can beused as a probe for detecting the DNA of the sialyltransferase-1; whichencode the polypeptides having an activity of the sialyltransferase-1;or which encode the polypeptides having antigenicity similar to that ofthe sialyltransferase-1, or DNAs or RNAs complementary thereto. Thehybridization referred to above may be performed under stringentconditions by a method which is conventionally used for hybridizing DNAor RNA with DNA, such as screening. For example, the conditions used inscreening DNA or the like include prehybridizing a target DNA in asolution containing 50% formamide, 5×SSPE (sodium chloride/sodiumphosphate/EDTA buffer), 5× Denhardt's solution, 0.5% SDS, and 50 μg/mlof denatured salmon sperm DNA, adding to the solution ³²P-labeled DNA ofthe present invention (for example, DNA having a nucleotide sequenceshown by SEQ ID NO: 1 or 7), hybridizing it at 42° C. for 16 hours, andthen washing it sequentially with 1×SSPE, 1% SDS, 0.1×SSPE, and 0.1% SDSat 55° C. Although generally hybridization is performed mostly under theabove-mentioned conditions, one skilled in the art can perform similarhybridization by changing the composition of each solution andconditions aiming at similar hybridization and, hence, the presentinvention is not limited to the above-described conditions as far as theconditions used enable one to obtain similar effects.

[0052] More specifically, the DNA of the present invention includes DNAshaving the whole nucleotide sequence shown by SEQ ID NO:1 or partialsequences thereof, which are preferred specific examples of these DNAsinclude a DNA having a nucleotide sequence of base numbers 202-1278 inthe nucleotide sequence shown by SEQ ID NO: 1 or base numbers 278-1363,365-1363, 389-1363 or 682-826 in the nucleotide sequence shown by SEQ IDNO: 7.

[0053] In the nucleotide sequence shown by SEQ ID NO: 1, the 5′-terminalportion of the open reading frame of cDNA of sialyltransferase-1contains three in-frame ATG codons. The nucleotide sequences around thethree ATG codons conserve each a purine base at the −3 position. Thissatisfies the Kozak's finding on efficient translation (Kozak, M.,(1986) Cell, 44, 283-292) so that it is possible that any of the ATGcodons functions as an initiation codon.

[0054] In the meantime, β-1,4-galactosyltransferase is known to containtwo in-frame ATG codons (Nakazawa, K. et al. (1988) J. Biochem., 104,165-168; Shaper, N. et al. (1988) J. Biol. Chem., 263, 10420-10428).Also, Shaper et al. showed that in the case ofβ-1,4-galactosyltransferase, translation starts at two sites, resultingin that the enzyme is synthesized in both longer and shorter forms.Further, Lopez et al. presented the evidence suggesting that the longerform preferentially targets membrane while the shorter form existsmainly in Golgi apparatus (Lopez, L. et al. (1991) J. Biol. Chem., 266,15984-15591). Similarly, in the case of the sialyltransferase, there isthe possibility that plural ATG codons serve as an initiation codon.This is not certain yet. However, no matter how ATG codon may be aninitiation codon, it is common that the polypeptide of theabove-mentioned sialyltransferase-1 is encoded. Therefore, DNAs havingnucleotide sequences starting with the second and third ATG codons,respectively are also embraced by the present invention. Specifically,the sialyltransferase-1 may have a region corresponding to amino acidnumbers 41-359 in the amino acid sequence of SEQ ID NO: 2 or amino acidnumbers 41-362 in the amino acid sequence of SEQ ID NO: 8.

[0055] From a single open reading frame starting with the first ATGcodon in the sequence shown by SEQ ID NO: 1 is deduced a protein whichconsists of 359 amino acid residues, has a molecular weight of 41,244Da, and contains four sites that can be an N-glycosylation site. Fromhydropathy plot prepared from this amino acid sequence, it can be seenthat there exists in the sequence a 14 residue-long continuous,remarkably hydrophobic part ranging from the 16th to 29th amino acidresidues counting from the N-terminal, which suggests that the proteinhas a transmembrane domain.

[0056] From a single open reading frame starting with the first ATGcodon in the sequence shown by SEQ ID NO: 7 is deduced a protein whichconsists of 362 amino acid residues, has a molecular weight of 41,754Da, and contains two sites that can be an N-glycosylation site. FIG. 5is a graph illustrating hydropathy plot prepared from this amino acidsequence. From FIG. 5 it can be seen that there exists in the sequence a14 residue-long continuous, remarkably hydrophobic part ranging from the16th to 29th amino acid residues counting from the N-terminal, whichsuggests that the protein has a transmembrane domain.

[0057] It will be readily understood by one skilled in the art that theDNA of the present invention also includes DNAs having differentnucleotide sequences by degeneracy of genetic codes.

[0058] Further, the DNA of the present invention include DNAs or RNAscomplementary to the DNA of the present invention. Furthermore, the DNAof the present invention may be of a single strand of only a codingstrand which encodes SAT-1 or a double strand of the single strand and aDNA or RNA strand having a nucleotide sequence complementary thereto.

[0059] Also, the DNA of the present invention may have a nucleotidesequence over the whole encoding region which encodes the whole peptideof SAT-1 or a nucleotide sequence encoding only a part of thepolypeptide of SAT-1.

[0060] Now, generally, mammal sialyltransferases are known to have highhomology in their amino acid sequence. The polypeptide which the DNA ofthe present invention encodes is expected to have a homology of about65% or more in the species The homology determined as a percentage ofnucleotides which are identical to corresponding nucleotides in thecoding region of SAT-1. Therefore, polypeptides having high homologywith the polypeptides encoded by the DNAs of the present inventionspecifically disclosed herein and DNAs encoding such polypeptides (suchas homologous or allelic variants) are also embraced by the presentinvention.

[0061] As described above, the polypeptide of SAT-1 has a transmembranedomain. The part of the polypeptide of SAT-1 that has lost the regionstarting from the N-terminal corresponding to the N-terminal inside themembrane and containing the region having the transmembrane domain isalso embraced by the present invention. As far as such a polypeptide hasan activity of SAT-1, the polypeptide is included in that contained inthe enzyme of the present invention. Such a polypeptide includes, forexample, an amino acid sequence of amino acids numbers 38 to 359 in theamino acid sequence shown by SEQ ID NO: 2, and amino acid numbers 30 to362, 38 to 362 and 41 to 362 in the amino acid sequence shown by SEQ IDNO: 8.

[0062] [2] Method of Producing the DNA of the Invention

[0063] Hereafter, a method of producing the DNA of the present inventionwill be explained in detail. As the amino acid sequence of thepolypeptide of SAT-1 has been clarified by the present invention, it ispossible to obtain the DNA by amplification from chromosomal DNA orm-RNA by a PCR method (polymerase chain reaction method) using anoligonucleotide primer prepared based on the amino acid sequence.Alternatively, the DNA of the present invention can also be produced byan expression cloning method, particularly the method which comprisesthe following steps.

[0064] (1) Cancer cells from mouse or human are treated with adifferentiation inducing agent to cause differentiation.

[0065] (2) A cDNA library is prepared from differentiated cancer cellsand is introduced into host cells.

[0066] (3) Host cells that have expressed ganglioside on the cellmembrane are screened.

[0067] (4) The screened host cells are sorted to enrich the library.

[0068] (5) The introduced gene is excised from the enriched library.

[0069] The whole length of cDNA of the above-mentioned SAT-1 is normallyselected by means of screening.

[0070] Hereafter, an example of the method of producing the DNA of thepresent invention will be explained more specifically.

[0071] (1) Differentiation Induction in Cancer Cells

[0072] Cancer cells are preferably anchorage independent cells frommouse or human. Such cancer cells include blood cell lymphoma andleukemia cells, which are preferred. As such cells are preferred, forexample, human-derived HL-60 (ATCC CCL-240), MOLT-4 (ATCC CRL-1582), andU937 (ATCC CRL-1593) and mouse-derived M1 (ATCC TIB-192) and B-16 (ATCCCRL-6322) and fresh myelogenous leukemia cells can also be used. Amongsuch cancer cells, most preferred are human-derived cells and HL-60cells are particular preferred since differentiation induction isreadily performed. Differentiation is induced by cultivating thecultivated cancer cell line for 20 hours or more, preferably for about24 to 48 hours, after adding a differentiation inducing agent to thecancer cell line. Cultivation may be performed under conditions that aresuited for the cells used. Usually, as general cell culture conditions,there can be used conditions of 5-7 vol % of CO₂ and 95-93 vol % of airat 37-38° C. As the differentiation inducing agent, there can be used,for example, phorbol ester (12-O-tetradecanoyl phorbol ester (TPA)etc.), dimethyl sulfoxide (DMSO), retinoic acid (RA), and1α,25-dihydroxyvitamin D₃ (1α,25(OH₂D₃)) and the like. Although thepresent invention is not limited to the use of a particular one,preferred among them is TPA since it has relatively uniformdifferentiation inducing activity toward many leukemia cell lines. WhenHL-60 is used as a cancer cell and TPA is used as a differentiationinducing agent, 48 hour cultivation in the presence of TPA in an amountof about 24 nM leads to the differentiation of HL-60 intomonocyte/macrophage like cells, showing morphological changes.

[0073] (2) Construction of cDNA from Differentiated Cancer Cells

[0074] 1) Preparation of RNA from Differentiated Cancer Cells

[0075] The cancer cells of which differentiation is induced in above (1)are collected by centrifugation preferably at 500 to 2,000×g and totalRNA is prepared from the cells by a known method, for example, aguanidine thiocyanate/CsCl method (Kingston, R. E., (1991) in CurrentProtocols in Molecular Biology, Suppl. 14, Unit 4.2, Green PublishingAssociates and Wiley Interscience, New York). From the total RNA thusobtained is purified poly(A)⁺RNA by oligo-(dT)cellulose columnchromatography or the like.

[0076] 2) Construction of cDNA from Poly(A)⁺ RNA

[0077] Reverse transcription PCR using the above-mentioned poly(A)⁺RNAas a template and also an oligonucleotide primers allows amplificationof cDNA derived from the cancer cells. PCR may be performed in the samemanner as a conventional method. Specific method thereof may be asfollows. Namely, a buffer solution (final volume 20 μl) containing 1 μlof poly(A)⁺RNA, 100 pmol of oligo-(dT), 100 pmol each of randomoligonucleotide primers, 500 μl each of 4 kinds of deoxyribonucleosidetriphosphates, 200 units of M-MLV reverse transcriptase (Gibco BRL), 1%Mdithiothreitol (DTT), 120 units of RNase (ribonuclease) inhibitor(manufactured by TAKARA SHUZO CO., LTD.) was incubated at 50° C. for 60minutes to synthesize a cDNA primary strand. Next, a reaction mixture(final volume 50 μl) containing 5 μl of the above-mentioned reversetranscriptase reaction mixture, 100 pmol each of random oligonucleotideprimers, 250 μM each of 4 kinds of deoxyribonucleoside triphosphates,and 1.25 units of Taq polymerase was incubated by repeating 35 cycles of95° C. for 1 minute, 46 to 62° C. for 1 minute, and 72° C. for 2minutes.

[0078] The cDNA of cancer cells thus obtained is made to be held by anexpression vector and introduced into host cells for screening the hostcells. As the host cells, there can be used any cells as far as they arecells of a mammalian-derived cell line which arelactosylceramide-positive. Examples of such include human Namalwa cells(Hosoi et al.: Cytotechnology, 1, 151 (1988)), Chinese hamster-derivedCHO cells (ATCC CCL61, etc.), monkey-derived COS cells (ATCC CRL1650,etc.), mouse-derived 3LL cells (Taniguchi, S., Shinshu University AgingAdaptation Research Center)) and so on. However, since the detection ofSAT-1 enzyme activity can be made easier in the present invention, thosecultivated cells which are further G_(M3)-negative are preferred.Examples of such cells include 3LL-HK46 cell (Inokuchi, J., (SeikagakuCorporation)), a mutant of 3LL cell, which is preferred. The expressionvector includes pCEV18 (Maruyama, K. (donated from Tokyo University,Medical Science Research Institute, now Tokyo Medical DentalUniversity), pCXN2 (Niwa, H., Yamamura, K. and Miyazaki, J. (Gene, 108,p193-200 (1991)), pPLAG-CMV-2 (manufactured by Eastman Kodak), pAGE107(Miyaji et al., Cytotechnology, 3, 133 (1990)), pAS3-3 (JP-A 2-227075),pAMoERC3Sc(JP-A 5-336963), pcD2 (Chen, C. et al., Mol. Cell. Biol., 7,2745-2752 (1987)) and the like and can be selected appropriately takinginto consideration the host cell to be used. For example, when 3LL-HK46is used as a host cell, it is preferred that pCEV18 be used as anexpression vector. Introduction, into a vector, of the PCR productprepared based on poly(A)⁺RNA of a cancer cell as described above isperformed by a method selected from known methods which is suited forthe vector to be used.

[0079] 3) Introduction of cDNA Library into a Host Cell

[0080] The cDNA library constructed by the above-mentioned method istransfected to host cells by a known technique. Specifically, there canbe cited, for example, an electroporation method (Miyaji, et al.,Cytotechnology, 3, 133 (1990)), a calcium phosphate method (JP-A2-227075), and a lipofection method (Philip, L. F. et al., Proc. Natl.Acad. Sci. USA, 84, 7413 (1987)), and selected suitably. However, theelectroporation method is preferred. Human a2-8 sialyltransferase is anenzyme synthesizing G_(D3) from G_(M3) and a cell to which a DNAencoding the enzyme expresses G_(D3) on its cell membrane. Detection ofthe G_(D3) can be easily conducted. When cDNA encoding SAT-1 isdetected, therefore, it is also possible and preferable to transfect, inadvance or simultaneously, to host cells, DNA encoding human α2-8sialyltransferase (JP-A 7-327678, etc.), in order to more preciselydetect the SAT-1 activity. Such a pretransfection or simultaneoustransfection is preferred. Therefore, when the cDNA library constructedby using pCEV18, for example, as a vector is introduced into 3LL-HK46cells having no G_(D3) synthesis pathway as a host cell, pCEV18 holdingthe cDNA library may be transfected to normal 3LL-HK46 cells directly orsimultaneously with the vector into which the cDNA of a2-8sialyltransferase has been introduced. Also, 3LL-ST28 cells originallyexpressing a2-8 sialyltransferase may be transfected with an eukaryoteexpression vector such as pCEV18, containing cDNA of the above-mentionedlibrary. The 3LL-ST28 cells is produced by introducing cDNA of a2-8sialyltransferase to 3LL-HK46 cells by using pCEV18.

[0081] (3) Detection of Host Cells Expressing Ganglioside Host cellsinto which cDNA library has been introduced are cultivated undergenerally used cell culture conditions. After at least 24 hours,preferably after 36 to 48 hours, from the introduction of cDNA, the hostcells are stained by immuno-staining using an anti-ganglioside antibodyor a lectin which bonds to ganglioside. The staining method usingantibodies is more accurate and preferred. For example, when 3LL-HK46cell is used as a host cell, the expressing cells are detected by usingan antibody which recognizes G_(M3) that has been expressed on the cellmembrane, for example, anti-G_(M3) monoclonal antibody M2590 (themonoclonal antibody which L612 (ATCC CRL10724)) produces: J. Biol.Chem., 260, 13328-13333 (1985)). The immuno-staining can be performed bya conventional method. When the above-mentioned 3LL-ST28 is used as ahost cell, for example, G_(D3) produced upon the introduction of the DNAof the present invention is detected. Immuno-staining for detectingG_(D3) may be performed by a conventional method generally used (JP-A2-327678). In this case, the primary antibody to be used is not limitedparticularly as far as it is an antibody which recognizes G_(D3).However, monoclonal antibodies are preferred and examples of whichinclude anti-G_(D3) monoclonal antibody R24 (monoclonal antibody which ahybridoma (ATCC HB8445) produces: Cancer Res., 49, p191-196 (1989)),which is preferred. Specifically, the immuno-staining method using theabove-mentioned generally employed antibodies is mentioned. Namely, thehost cells (1×10⁵ cells) after the above-mentioned cultivation arewashed about 2 or 3 times by centrifugation in a BSA solution (0.1% BSAPBS(+)) and the cells are suspended in 100 μl of the BSA solutioncontaining the primary antibody. After allowing the suspension to reactunder ice cooling for 30 minutes, the cells are washed with theabove-mentioned BSA solution 2 times or so. Further, in 100 μl of a BSAsolution containing 1 μl of FITC-labeled secondary antibody against theprimary antibody, the cells are left to stand for 30 minutes under icecooling for reaction. The cells are washed with a BSA solution once andthose cells which show strong fluorescence are detected by using a flowcytometer (FACScalibur: manufactured by Becton Dickinson). Cells showingstrong fluorescence, for example, 5% of the total cells, are selected bya cell sorter and plasmid DNA is extracted therefrom. The extraction ofplasmid DNA from the host cells is performed by a conventional method.

[0082] (4) Sorting of SAT-1 cDNA and Obtaining of cDNA

[0083] The plasmid DNA obtained by the above-described operation istransfected to a suitable host cell line and the procedure ofimmuno-staining with the anti-G_(M3) antibody and recovery of strongfluorescence-showing cells in an amount of 5% of the total cells byusing a flow cytometer, for example, is repeated twice or more to enrichthe target cDNA by sorting. The host cell used for the sorting ispreferably cultivated mammalian cells, of which 3LL-HK46 is particularlypreferred. The vector to be used is not limited particularly and anyexpression vectors for mammalian cells may be used, but pCEV18 ispreferred The above-mentioned vector holding the target cDNA enriched bysorting is transfected to mammalian-derived cultivated cells lackingG_(D3) synthesis pathway, such as 3LL-HK46, simultaneously with theexpression vector made by introducing a human a2-8 sialyltransferasecDNA into an expression vector for mammalian cells, such as pBKCMV(manufactured by STRATAGENE CO.) and detection by immuno-staining and aflow cytometer is conducted in the same manner as described above toobtain cells that show strong fluorescence in an amount of 5% of thetotal cells From these cells is extracted plasmid DNA by a conventionalmethod. The cDNA excised by a conventional method from the plasmid DNAis used to transform E. coli DH10B (manufactured by GIBCO CO.)therewith, and the transfected E. coli cells are inoculated so that 100colonies per well can be formed, followed by sib selection to finallyobtain a clone containing an insert of about 2 kbp.

[0084] (5) Determination of the Nucleotide Sequence of cDNA EncodingSAT-1

[0085] The nucleotide sequence of the cDNA obtained as described above,as is or after subcloning in a suitable plasmid such as pCRII, isdetermined by a conventional method.

[0086] The nucleotide sequence of the mouse SAT-1-encoding cDNAdetermined as described above and amino acid sequence deduced from thenucleotide sequence are shown by SEQ ID NO: 1 and the amino acidsequence alone is shown by SEQ ID NO: 2.

[0087] The nucleotide sequence of the human SAT-1-encoding cDNAdetermined as described above and amino acid sequence deduced from thenucleotide sequence are shown by SEQ ID NO: 7 and the amino acidsequence alone is shown by SEQ ID NO: 8.

[0088] Further, the DNA encoding the polypeptide of SAT-1 which lacks atransmembrane domain, i.e., which is in the form of solubilized proteincan be obtained as follows. Namely, based on the nucleotide sequenceshown by SEQ ID NO: 1 or 7 is prepared a primer selected to have atruncated form at the N-terminal side of the polypeptide of the enzyme,and the target DNA is amplified by a PCR method using the cDNA of clonedSAT-1 as a template. For example, when a DNA encoding the polypeptide ofa truncated form that lacks 37 amino acid residues at the N-terminal isto be obtained, an oligonucleotide primer is synthesized based on thenucleotide sequence existing at the 3′- and 5′-terminals of the targetnucleotide sequence, for example. An oligonucleotide primers havingnucleotide sequences are shown by SEQ ID NO: 3 and SEQ ID NO: 4 for thenucleotide sequence shown by SEQ ID NO: 1, SEQ ID NO: 9 and SEQ ID NO:10 for the nucleotide sequence shown by SEQ ID NO: 7, respectively, forexample, may be used as 5′- and 3′-primers, respectively, in order toperform PCR. Then, the target DNA can be obtained from the amplified PCRproduct, after purification, if desired.

[0089] [3] SAT-1 Polypeptide Encoded by the Nucleotide Sequence of theDNA of the Present Invention

[0090] The present invention provides SAT-1 polypeptide encoded by theDNA of the present invention. The polypeptide may be single or fusedwith one or more other polypeptides. The polypeptide may also lack atransmembrane domain.

[0091] The polypeptide may be with or without a sugar chain. The kind ofsugar chain is not limited particularly.

[0092] Such a polypeptide can be obtained by, for example, theproduction method as described below Determination of presence orabsence of the above-mentioned activity or function can be practiced bychanging the cDNA to be introduced into host cells and the substrate forthe enzyme in the assay of enzyme activity as described in JP-A 7-327678and can be performed with ease by one skilled in the art based on, forexample, the method described herein specifically.

[0093] [4] Production Method for SAT-1 or Polypeptide Thereof Utilizingthe DNA of the Present Invention

[0094] The SAT-1 or the polypeptide thereof can be produced bycultivating cells transformed with the above-mentioned DNA of thepresent invention in a suitable medium, to produce and accumulate in theculture the SAT-1 or the polypeptide thereof encoded by the DNA of thepresent invention, and collecting the SAT-1 or the polypeptide thereoffrom the culture.

[0095] The cells transformed with the DNA of the present invention canbe obtained by inserting a fragment of the DNA of the present inventioninto a known expression vector to construct a recombinant plasmid andconducting transformation with the recombinant plasmid. The presentinvention also provides a recombinant vector, that is, a recombinantplasmid comprising the DNA of the present invention; a transformant intowhich the DNA of the present invention is introduced, and in which theDNA can be expressed (for example, a transformant comprising therecombinant vector) which can be used for production of the enzyme ofthe present invention.

[0096] Examples of the cells which can be used include prokaryotic cellssuch as E. coli and eukaryotic cells such as mammalian cells. Whenprokaryotic cells such as E. coli are used, there occurs no addition ofsugar chain to the SAT-1 polypeptide to be produced by the expression ofthe DNA of the present invention and, hence, pure SAT-1 polypeptide canbe obtained. On the other hand, when eukaryotic cells such as mammaliancells are used, addition of sugar chain(s) to the SAT-1 polypeptideproduced by the expression of the DNA of the present invention occurs.Therefore, polypeptides can be obtained in the same form as ordinarySAT-1 which contains also a sugar chain.

[0097] In this production method, a host-vector-system usually used inthe production of proteins may be used. While it is preferred to use acombination of mammalian-derived cultivated cell, such as 3LL-HK46 cell,3LL-ST28 cell or COS-1 cell, and an expression vector for mammaliancells, such as pCEV18, pME18S (Maruyama et al., Med. Immunol., 20, 27(1990)), the present invention is not limited thereto. The medium andcultivation conditions may be selected suitably depending on the hostcell to be used.

[0098] While the DNA of the present invention may be expressed over thewhole length thereof, it may be expressed as a fused polypeptide withanother polypeptide. Also, a part of the DNA of the present inventionmay be expressed as a partial polypeptide.

[0099] A specific example of construction of recombinant plasmid whichexpresses the above-mentioned fused polypeptide is by the followingmethod. Namely, the DNA of the present invention is incorporated into avector constructed so that a gene introduced-into a plasmid such aspGIR201protA (Kitagawa, H. and Paulson, J. C., J. Biol. Chem., 269,1394-1401 (1994)) can be expressed as a fused protein by a conventionalmethod to construct a vector having genes for plural proteins on thesame reading frame. Then, from the vector is excised NheI fragment,which encodes a fused protein, and the fragment is ligated to a suitablevector such as pCEV18 by the same operation as described above.

[0100] The SAT-1 or the polypeptide thereof of the present invention canbe collected from the culture by a known purification method forpolypeptides. Specifically, there can be used affinity chromatographyusing a Sepharose column to which lactosylceramide or CMP-sialic acid,for example is bonded. When the DNA of the present invention isexpressed as a fused polypeptide, the culture of the host cell can besubjected to affinity chromatography using a column to which a substanceis bonded having high affinity for the polypeptide fused with SAT-1,such as antibody, thereby purifying the fused polypeptide. A linkerhaving an amino acid sequence which a specified proteolytic enzyme canrecognize and cleave may be incorporated in advance between the SAT-1and the other polypeptide in the fused polypeptide. This allows thecleavage to occur at the linker site of the fused polypeptide afterpurification thereof so that SAT-1. The combination of the specifiedproteolytic enzyme and the specified sequence which the enzymerecognizes is, for example, a combination of signal peptidase which actsupon the synthesis of proinsulin and signal peptide of insulin. Theabove-mentioned culture includes a medium and cells in the medium.

[0101] The activity of sialyltransferase can be assayed by changing thesubstrate for the enzyme in a conventional assay method for assayinggeneral ganglioside synthesis (JP-A 7-327678). For example, a suitableamount of the culture or the enzyme purified by the above-describedmethod is added to a reaction mixture containing 100 mM sodiumcacodylate, 10 mM manganese chloride, 0.2 mM CMP-radioactivesubstance-labeled sialic acid, 0.4 mM lactosylceramide, and 0.3% TritonCF-54. The mixture is adjusted to pH 6.5 and incubated at 37° C. for 2hours and the reaction product is developed by a conventional thin layerchromatography and the enzyme activity is determined by using FujixBAS2000 Bio Imaging Analyzer (manufactured by Fuji Photo Film Co.,Ltd.).

EXAMPLES

[0102] The present invention will be described in further detail byexamples. However, the present invention is not limited thereto withoutexceeding the object of the present invention.

Example 1

[0103] (1) Differentiation Induction of B-16 Cell and Construction ofcDNA

[0104] Mouse melanoma B-16 cells were cultivated in RPMI-1640(manufactured by NISSUI PHARM. CO.) containing 24 nM TPA under theconditions of 5 vol % CO₂ and 95 vol % air at 37° C. for 48 hours toinduce differentiation. The cultivated cells were collected bycentrifugation at 1000×g and total RNA was prepared from the collectedcells by guanidine thiocyanate-acid-phenol-chloroform method (AGPCmethod). From differentiated 5×10⁶ cells was obtained about 40 μg ofRNA. From the RNA, poly(A)⁺RNA was purified by oligo-(dT) cellulosecolumn chromatography.

[0105] The poly(A)⁺RNA was used as a template for reverse transcriptionreaction to construct a primary strand of DNA, and the DNA in turn wasused for synthesizing double-stranded cDNA (Gubber, V. and Hoffman, B.J., Gene, 25, 283 (1983)).

[0106] To the double-stranded cDNA was ligated a restriction enzymeBSTX1 adapter and the ligate was introduced into the BSTX1 site ofpCEV18 to construct a cDNA library.

[0107] (2) Transfection of cDNA to 3LL-HK46 Cells

[0108] The above-mentioned cDNA library was introduced into 3LL-HK46cells by using an electroporation method and the transfected cells werecultivated for 48 hours under the conditions of 5 vol % CO₂ and 95 vol %air at 37° C.

[0109] (3) Detection of Host Cells Expressing Ganglioside andPreparation of cDNA

[0110] The 3LL-HK46 cells after the cultivation were immuno-stained withM2590, anti-G_(M3) antibody, and with FITC-labeled rabbit anti-murineIgG antibody. The stained cells were passed through a flow cytometer(FACScalibur) to detect fluorescence-positive cells. 5% of the cells onthe positive side were collected and plasmid DNA was prepared therefrom.Then, the procedures of introduction of cDNA into 3LL-HK46 cells byelectroporation, 48-hour cultivation of the transfected cells,immuno-staining, and detection and collection by using a flow cytometerwere further repeated twice.

[0111] The plasmids finally obtained by this method were introduced into3LL-HK46 cells together with pBKCMV G_(D3) (a plasmid obtained byintroducing the cDNA of human α2-8 sialyltransferase (G_(D3) synthase)to pBKCMV plasmid vector manufactured by STRATAGENE CO.). Aftercultivating them for 48 hours, the resulting cells were immuno-stainedwith R24, anti-G_(D3) antibody, and with FITC-labeled rabbit anti-murineIgG antibody, and 5% of the total cells which show strong fluorescencewere detected by a flow cytometer and collected.

[0112] From these cells was prepared plasmid DNA, which then wastransfected to E. coli DH10B (manufactured by GIBCO) by electroporation.After repeating the transfection and screening with ampicillin twice,positive colonies were dispensed to each well in a 96-well microplate inan amount of 100 colonies per well. Nine (9) microplates were inoculatedwith the transfected cells and only one well was selected by sibselection. Then, 2,400 colonies derived from this single well wereextended to twenty five (25) 96-well microplates in a population of 1colony per well and further sib selection gave rise to a positive clone(pCEVmS1). The pCEVmS1 thus obtained was expressed in 3LL-HK46 cellstemporarily and flow cytometry analysis was performed using anti-G_(M3)antibody (M2590) in the same manner as described above. 3LL-HK46 cellstemporarily expressing pCEV18, as a control, did not express G_(M3) onthe cell membrane whereas 3LL-HK46 cells temporarily expressing pCEVmS1expressed G_(M3) on the cell membrane and fluorescence was detected.

[0113] (4) Determination of Nucleotide Sequence

[0114] The nucleotide sequence of double-stranded DNA of pCEVmS1 wasdetermined by a dideoxy chain termination method using an autocyclesequencing kit (manufactured by PHARMACIA CO.) and Pharmacia A.L.F. DNAsequencer (manufactured by PHARMACIA CO.). The nucleotide sequence thusdetermined and an amino acid sequence deduced therefrom are shown by SEQID NO: 1 and the amino acid sequence alone is shown by SEQ ID NO. 2. ThecDNA insert mS1 which is contained in pCEVmS1 is of about 2.1 kbp and isrevealed to encode a protein (molecular weight 41,244 Da containing 359amino acid residues starting with a nucleotide at 202 position as atranslation initiation point. FIG. 1 is a schematic view whichillustrates the structure expected from the amino acid sequence. As aresult of hydropathy plot analysis, the amino acid sequence was revealedto correspond to a type-2 membrane protein in which the transmembranedomain (TM in FIG. 1) exists in the region of the 16th to 29th aminoacid residues on the N-terminals. Search of this sequence with gene database in GenBank showed no high homology with any of the data therein.However, with regard to the sialylmotifs (L and S) in thesialyltransferase homologous region existing in the central part andC-terminal region of the sequence for sialyltransferase, relatively highhomology was recognized although some substitution was observed (FIG.2). The sialyltransferases used for comparison were eleven (11) species,i.e., h2,3ST (JP-A 5-336963), rSTX (J. Biol. Chem., 268, 11504-11507(1993)), rST3N-1 (J. Biol. Chem., 267, 21011-21019 (1992)), hST3N-2 (J.Biol. Chen., 268, 22782-22787 (1993)), pST30-1 (J. Biol. Chem., 276,21004-21010 (1992)), mST30-2 (Eur. J Biochem., 216, 377-385 (1993)),mST4′ (NCBI Seq. ID 558532), hSAT4(a) (Gycbiology, 5, 319-325 (1995)),hST6N (Nuc. Acids Res., 18, 667 (1990)), rST6N (J. Biol. Chem., 262,17735-17743 (1987)), h2,8ST (JP-A 7-327678). The results suggest thatSAT-1 which is encoded by the insert mS1 in pCEVmS1 belongs to thesialyltransferase family. Further, the amino acid sequence indicatesexistence of four consensus sequences of the N-glycosylation site (Δ inFIG. 1), whereas the two sites thereof on the N-terminal side exist nearthe transmembrane domain and in the sialylmotifs so that these twoN-terminal side sites could be less N-glycosylated as compared with thetwo sites on the C-terminal side.

[0115] (5) G_(M3) Synthesis in Cells Expressing SAT-1 cDNA

[0116] pCEVmS1 obtained by incorporating the above-mentionedSAT-1-encoding cDNA (mSAT-1 cDNA) into expression vector pCEV18 wastransfected to 3LL-HK46 cells by an electroporation method and theG_(M3) synthase activity of the cells after 48-hour cultivation wasassayed by the following method. Namely, 20 μl of a reaction mixture (pH6.5) containing 0.1 mM CMP-(C)-sialic acid (2×10³ CPM), 0.4 mMlactosylceramide, 0.3% (W/V) Triton CF-54, 10 mM MgCl₂, 100 mM sodiumcacodylate, 150 μg of the homogenate of 3LL-HK46 cells to which pCEVmS1was incorporated, and 1 mM sialidase inhibitor(2,3-dehydro-2-deoxy-N-acetylsialic acid (2,3-dehydro-2-deoxy-NeuAc,manufactured by BOEHRINGER MANNHEIM GMBH) was incubated at 37° C. for0.2 hours and then 10 μl of methanol was added thereto to stop thereaction. 8 μl of the reaction mixture was charged on a C18 reversedphase thin layer chromatography plate (RP-18W HPTLC plate, manufacturedby MERCK CO.) and developed with water for 10 minutes. Radioactivesubstance-labeled reaction products were scrubbed from the originalpoint and G_(M3) was collected therefrom by extraction with 300 μl ofchloroform/methanol (1:1, V/V). After the extracts were concentrated todryness, they were charged on a 60HPTLC plate (manufactured by MERCKCO.) for silica gel thin layer chromatography. After development withchloroform/methanol/0.5% aqueous CaCl₂ solution (55:45:10:, V/V/v), thelayer was treated with orcinol sulfate to develop color and measured ofradioactivity incorporated into ganglioside using Fujix BAS2000 BioImaging Analyzer (manufactured by FUJI PHOTO FILM CO., LTD.). Theresults revealed uptake of ¹⁴C by ganglioside G_(M3) and G_(M3)synthesis by SAT-1 was detected in the SAT-1 cDNA-transfected cells.

[0117] The G_(M3) synthase activity was high at pH 6.0 to 7.0,particularly at around pH 6.5 and increased at least 1.5 times in thepresence of 10 mM of Mn²⁺.

Example 2

[0118] (1) Differentiation Induction of HL-60 Cell and Construction ofcDNA

[0119] HL-60 cells (2×10⁵ to 3×10⁵ cells/ml) were cultivated inRPMI-1640 (manufactured by NISSUI PHARM. CO.) containing 24 nM TPA and10% fetal calf serum under the conditions of 5 volt CO₂ and 95 vol % airat 37° C. for 48 hours to induce differentiation. From the cells,poly(A)⁺RNA was isolated using a Fast Track mRNA isolation kit(Invitrogen).

[0120] The poly(A)⁺RNA was used as a template for reverse transcriptionreaction to construct a primary strand of DNA, and the DNA in turn wasused for synthesizing double-stranded cDNA (Gubber, V. and Hoffman, B.J., Gene, 25, 283 (1983)).

[0121] To the double-stranded cDNA was ligated a restriction enzymeBSTX1 adapter and the ligate was introduced into the BSTX1 site ofpCEV18 to construct a cDNA library. The cDNA library was divided intoeight parts, and each part was amplified separately in Escherichia coliDH10B (Life Technologies, Inc.). The amplified cDNA was purified withQiagen Tip (Qiagen).

[0122] (2) Transfection of cDNA to 3LL-HK46 Cells

[0123] The above-mentioned cDNA library (100 μg of the plasmid DNA) wasintroduced into 5×10⁶ 3LL-HK46 cells by using an electroporation method(180 V, 600 μF) and the transfected cells were cultivated for 48 hoursunder the conditions of 5 vol % CO₂ and 95 vol % air at 37° C.

[0124] (3) Detection of Host Cells Expressing Ganglioside andPreparation of cDNA

[0125] The 3LL-HK46 cells after the cultivation were collected andwashed with PBS(−). Then, the cells were reacted with anti-G_(M3)antibody M2590 for 30 min on ice, and immuno-stained withFITC-conjugated rabbit anti-mouse IgG monoclonal antibody for 30 min onice. The stained cells were passed through a flow cytometer(FAcscalibur) to detect fluorescence-positive cells 5% of the cells onthe positive side were collected with an EPICS Elite ESP cell sorter(Coulter), and plasmid DNA was prepared therefrom. Then, the proceduresof introduction of cDNA into 3LL-HK46 cells by electroporation, 48-hourcultivation of the transfected cells, immuno-staining, and detection andcollection by using a flow cytometer were further repeated twice.

[0126] The plasmids finally obtained by this method were introduced into5×10⁶ 3LL-HK46 cells together with pBKCMV G_(D3) (a plasmid obtained byintroducing the cDNA of human α2-8 sialyltransferase (G_(D3) synthase)to pBKCMV plasmid vector manufactured by STRATAGENE CO.). Aftercultivating them for 48 hours, the resulting cells were immuno-stainedwith anti-G_(D3) antibody R24, and with FITC-conjugated rabbitanti-mouse IgG antibody. Cells which show strong fluorescence weredetected by a flow cytometer and 0.6% of the cells on the strongfluorescent side were collected with a FACS Vantage cell sorter (BectonDickinson).

[0127] From these cells was prepared plasmid DNA, which then wastransfected to E. coli DH10B by electroporation. After repeating thetransfection and screening with ampicillin twice, positive colonies weredispensed to each well in a 96-well microplate in an amount of 100colonies per well. Nine (9) microplates were inoculated with thetransfected cells and only one well was selected by sib selection. Then,2,400 colonies derived from this single well were extended to twentyfive (25) 96-well microplates in a population of 1 colony per well andfurther sib selection gave rise to a positive clone (pCEV4C7).

[0128] In particular, when 3LL-ST28 cells were used as a host cell, notless than 3 times fluorescence intensity was obtained compared with acase in which 3LL-HK46 cells were co-transfected with a plasmid DNAcomprising the DNA of the present invention and pBKCMVGD3. Therefore, inthe above-mentioned sib selection, co-transfection was not used and3LL-ST28 cells were used as a host cell.

[0129] (4) Determination of Nucleotide Sequence

[0130] The nucleotide sequence of double-stranded DNA of pCEV4C7 wasdetermined by a dideoxy chain termination method using an autocyclesequencing kit (manufactured by PHARMACIA CO.) and Pharmacia A.L.F. DNAsequencer (manufactured by PHARMACIA Co.). The nucleotide sequence thusdetermined and an amino acid sequence deduced therefrom are shown by SEQID NO: 7 and the amino acid sequence alone is shown by SEQ ID NO: B. ThecDNA insert 4C7 which is contained in pCEV4C7 is of about 2,359 bp andis revealed to encode a protein (molecular weight 41,754 Da containing362 amino acid residues starting with a nucleotide at 278 position as atranslation initiation point. FIG. 3 is a schematic view whichillustrates the structure expected from the amino acid sequence. As aresult of hydropathy plot analysis, the amino acid sequence was revealedto correspond to a type-2 membrane protein in which the transmembranedomain (TM in FIG. 3) exists in the region of the 16th to 29th aminoacid residues on the N-terminals. Search of this sequence with gene database in GenBank showed no high homology with any of the data therein.However, with regard to the sialylmotifs (L and S) in thesialyltransferase homologous region existing in the central part andC-terminal region of the sequence for sialyltransferase, relatively highhomology was recognized although some substitution was observed (FIG.4). The sialyltransferases used for comparison were eleven (11) species,i.e., ST3N-1 (Biochem. Biophys. Res. Commun., 194, 375-382, 1993) ST3N-2(J. Biol. Chem., 268, 22782-22787, 1993), ST30-1 (J. Biol. Chem., 269,17872-17878, 1994), ST30-2 (Eur. J. Biochem., 247, 558-566, 1997), SThM(GenBank™ database, accession number U14550), ST6N (J. Exp. Med., 172,641-643, 1990), SAT-II (Proc. Natl. Acad. Sci. U.S.A., 91, 7952-7956,1994), STX (J. Biol. Chem., 270, 22685-22688, 1995), ST8SiaIII (GenBank™database, accession number AF004668), PST-1 (Proc. Natl. Acad. Sci.U.S.A., 92, 7031-7035, 1995), ST8SiaV (Biochem. Biophys. Res. Commun.,235, 327-330, 1997). The results suggest that SAT-1 which is encoded bythe insert 4C7 in pCEV4C7 belongs to the sialyltransferase family. InSAT-1 encoded by the DNA, a characteristic amino acid substitution(substitution of histidine for aspartic acid):at 177th amino acid in thesialylmotif L, compared with other sialyltransferases. Further, theamino acid sequence indicates existence of two consensus sequences ofthe N-glycosylation site (A in FIG. 3).

[0131] (5) G_(M3) synthesis in cells expressing SAT-1 cDNA

[0132] pCEV4C7 obtained by incorporating the above-mentionedSAT-1-encoding cDNA (4C7) into expression vector pCEV18 was transfectedto 3LL-HK46 cells and 3LL-ST28 cells by an electroporation method andthe G_(M3) synthase activity of the cells after 48-hour cultivation wasassayed by the following method. As controls, pCEV18 was transfected to3LL-HK46 cells and 3LL-ST28 cells by the same method. 20 μl of areaction mixture (pH 6.5) containing 0.1 mM CMP-(¹⁴C)-sialic acid (2×10³CPM), 0.4 mM lactosylceramide, 0.3% (W/V) Triton CF-54, 10 mM MgCl₂, 100mM sodium cacodylate, 150 μg of the homogenate of host cells to whichpCEV4C7 (or control plasmid) was incorporated, and 1 mM sialidaseinhibitor (2,3-dehydro-2-deoxy-N-acetylsialic acid(2,3-dehydro-2-deoxy-NeuAc, manufactured by BOEHRINGER MANNHEIM GMBH)was incubated at 37° C. for 2 hours and lipid components were purifiedon SepPak C18 column (manufactured by MERCK CO.). The purified materialwas evaporated to dryness and charged on a 60HPTLC plate (manufacturedby MERCK CO.) for silica gel thin layer chromatography. Afterdevelopment with chloroform/methanol/0.5% aqueous CaCl₂ solution(55:45:10:, V/v/v), the layer was treated with orcinol sulfate todevelop color and measured of radioactivity incorporated intoganglioside using Fujix BAS2000 Bio Imaging Analyzer (manufactured byFUJI PHOTO FILM CO., LTD.). The results revealed uptake of ¹⁴C byganglioside G_(M3) and G_(M3) synthesis by SAT-1 was detected in thepCEV4C7-transfected cells. This indicated that G_(M3) synthesis by SAT-1occurred.

[0133] The G_(M3) synthase activity was high at pH 6.0 to 7.0,particularly at around pH 6.5 and increased at least 1.5 times in thepresence of 10 mM of Mn²⁺.

[0134] The 3LL-HK46 cells and the 3LL-ST28 cells were transfected withabove-mentioned pCEV4C7. Forty-two hours after transfection, the cellswere subjected to fluoroimmuno-staining (anti-G_(M3) antibody M2590 andanti-G_(M3) antibody R24 were used as a primary antibody for 3LL-HK46cells and 3LL-ST28 cells, respectively, and FITC-conjugated anti-mouseIgM antibody or IgG antibody was used as secondary antibody) anddistributions of stained cells were determined by flowcytometry. Ascontrols, each of host cells which were transfected with pCEV18 andimmuno-stained was used. The results are shown in FIG. 6. (a) and (b)are 3LL-ST28 cells, and (c) and (d) are 3LL-HK46 cells. (a) and (c) aretransfected with pCEV18 (controls) and (b) and (d) are transfected withpCEV4C7. It is clear that the 3LL-ST28 transfected with plasmid DNAharboring the DNA of the present invention is remarkably stainable. Thedifficulty of detection of G_(M3) by this method in 3LL-HK46 cellssuggests that localization or the like of G_(M3) on the cell surface isdifferent between cell lines.

[0135] (6) Expression of SAT-1 in Tissues

[0136] Expression of SAT-1 in tissues or the like was determined byNorthern blot analysis. Namely, MTN blos (Clontech) were used, and a2,066-bp fragment which was excised from the pCEV4C7 with EcoRI wasprepared by agarose gel electrophoresis, and radiolabeled with[α-³²P]dCTP by a usual method, to prepare a radiolabeled probe. Aradiolabeled human glyceraldehyde-3-phosphate dehydrogenase gene probealso was used as an internal control for normalizing the amount RNA ineach sample. The analysis showed that SAT-1 highly expressed in brain,placenta, skeletal muscle and prostate, whereas it was very weaklyexpressed in liver, kidney, pancreas and colon. In brain, placenta,lung, skeletal muscle, spleen and peripheral blood leukocytes, a minorband of 7 kilobases was detected.

[0137] To characterize in more detail the expression of SAT-1 in brain,Northern blotting analysis of cerebellum, cerebral cortex, medulla,occipital pole, frontal lobe, temporal lobe, putamen and spinal cord ofbrain was performed with the same probe. The analysis showed that SAT-1relatively highly expressed over the whole brain, but slightly elevatedexpression was observed in cerebral cortex, temporal lobe and putamen.

1 12 1 2121 DNA Mus musculus CDS 202..1278 misc_feature 247..288transmembrane domain 1 cccgggctgg cggcttgcca gcgctccctc cctagcatgcacacagaggc ggtgggcggc 60 gcggcgcgga ggccccagaa gctgcgaagc caagcagcggcacctgcctg ccgagcaatg 120 ccaagtgagt tcacctctgc aaagctgaga agtgattgctcaaggacctc cctgcaatgg 180 tacacccgaa cccagcacaa g atg aga aga ccc agcttg tta ata aaa gac 231 Met Arg Arg Pro Ser Leu Leu Ile Lys Asp 1 5 10atc tgc aag tgc acg ttg gtt gca ttt gga gtc tgg ctc ctg tac atc 279 IleCys Lys Cys Thr Leu Val Ala Phe Gly Val Trp Leu Leu Tyr Ile 15 20 25 ctcatt ttg aat tac acc gct gaa gaa tgt gac atg aaa aga atg cac 327 Leu IleLeu Asn Tyr Thr Ala Glu Glu Cys Asp Met Lys Arg Met His 30 35 40 tat gtggac cct gac cgg ata aag aga gct cag agc tat gct cag gaa 375 Tyr Val AspPro Asp Arg Ile Lys Arg Ala Gln Ser Tyr Ala Gln Glu 45 50 55 gtc ttg cagaag gaa tgt cgg ccc agg tac gcg aag acg gct atg gct 423 Val Leu Gln LysGlu Cys Arg Pro Arg Tyr Ala Lys Thr Ala Met Ala 60 65 70 ctg tta ttt gaggac agg tac agc atc aac ttg gag cct ttt gtg cag 471 Leu Leu Phe Glu AspArg Tyr Ser Ile Asn Leu Glu Pro Phe Val Gln 75 80 85 90 aag gtc ccc acggcc agt gaa gct gag ctc aag tat gac ccg cct ttt 519 Lys Val Pro Thr AlaSer Glu Ala Glu Leu Lys Tyr Asp Pro Pro Phe 95 100 105 gga ttc cgg aagttc tcc agt aaa gtc cag agc ctc ttg gat atg ctg 567 Gly Phe Arg Lys PheSer Ser Lys Val Gln Ser Leu Leu Asp Met Leu 110 115 120 ccc gaa cat gacttt cct gaa cac ttg aga gcc aag gcc tgc aag cgc 615 Pro Glu His Asp PhePro Glu His Leu Arg Ala Lys Ala Cys Lys Arg 125 130 135 tgt gtg gtt gttggg aac ggg ggc atc ctg cac gga cta gag ctg ggt 663 Cys Val Val Val GlyAsn Gly Gly Ile Leu His Gly Leu Glu Leu Gly 140 145 150 cac gcc ctc aaccag ttc gat gtg gta ata agg ttg aac agt gcg cca 711 His Ala Leu Asn GlnPhe Asp Val Val Ile Arg Leu Asn Ser Ala Pro 155 160 165 170 gtt gag ggttac tct gaa cac gtt ggg aat aaa act act ata agg atg 759 Val Glu Gly TyrSer Glu His Val Gly Asn Lys Thr Thr Ile Arg Met 175 180 185 act tac ccagag ggt gcg cca ctg tcg gac gtt gaa tac tac gcc aat 807 Thr Tyr Pro GluGly Ala Pro Leu Ser Asp Val Glu Tyr Tyr Ala Asn 190 195 200 gat ttg ttcgtt act gtt tta ttt aag agt gtt gat ttc aag tgg ctt 855 Asp Leu Phe ValThr Val Leu Phe Lys Ser Val Asp Phe Lys Trp Leu 205 210 215 caa gca atggta aaa aat gaa agc ctg ccc ttt tgg gtt cgc ctc ttc 903 Gln Ala Met ValLys Asn Glu Ser Leu Pro Phe Trp Val Arg Leu Phe 220 225 230 ttt tgg aagcaa gtg gca gaa aaa gtc cca ctc cag cca aag cac ttc 951 Phe Trp Lys GlnVal Ala Glu Lys Val Pro Leu Gln Pro Lys His Phe 235 240 245 250 agg attttg aac cca gtt atc atc aaa gaa act gcc ttc gac atc ctt 999 Arg Ile LeuAsn Pro Val Ile Ile Lys Glu Thr Ala Phe Asp Ile Leu 255 260 265 cag tactca gag cct cag tca aga ttc tgg ggc cat gat aag aac atc 1047 Gln Tyr SerGlu Pro Gln Ser Arg Phe Trp Gly His Asp Lys Asn Ile 270 275 280 ccc acgatc ggc gtc att gcc gtt gtc ttg gct aca cat ctg tgt gat 1095 Pro Thr IleGly Val Ile Ala Val Val Leu Ala Thr His Leu Cys Asp 285 290 295 gaa gtcagc ctg gca ggc ttt ggc tac gac ctc agt caa ccc agg acc 1143 Glu Val SerLeu Ala Gly Phe Gly Tyr Asp Leu Ser Gln Pro Arg Thr 300 305 310 cct ctgcac tac ttt gac agt cag tgc atg ggc gcc atg cac tgg cag 1191 Pro Leu HisTyr Phe Asp Ser Gln Cys Met Gly Ala Met His Trp Gln 315 320 325 330 gtcatg cac aat gtg acc aca gag acc aag ttc ctc ctg aag ctc ctc 1239 Val MetHis Asn Val Thr Thr Glu Thr Lys Phe Leu Leu Lys Leu Leu 335 340 345 aaggag ggc gtg gtg gag gac ctc agc ggc ggc atc cac tgagaactcg 1288 Lys GluGly Val Val Glu Asp Leu Ser Gly Gly Ile His 350 355 gaacacggcaaacctcaccc agcaccgcag ctgagagcgt ggtgagcagc ctccacaggg 1348 acttcaccctgcagctgctt cgatgtgcag ctagtgtttt caaactccac atttttttta 1408 aaaaaggaaaagaaagaaca acagcaacaa caaaagctct gctctgtgca cctcttcgtc 1468 ctatttatttgaagtcagtg ttggattttg cacagttttg taagttaatc ttaagaatgg 1528 gattggaaggacttttcaaa gagaattgta tagtttattg ttttttaagg aagtaattta 1588 atttgcagaaactgtacaca cgtactctgc tcaggtgttg aggtgggagg agaggggctt 1648 ctggcccctggatgatggct gtgatgcccg atactggggt ctgctgctct gtttggtaga 1708 actgatggcagagaaacttc ctgcctccag gataaagggc ttactcatca cctctggcag 1768 ctgctagacaagttcataac ccctttctgc tagtccatct gccagctggc tcgcaggact 1828 caggcagggcagctgtcccg gaggctgctg gttggtgagc cactgtcagc tgagcgccgt 1888 gatgttgccccagggtggaa gaagccacac ttcctacact gtcagggcac ttttaaactt 1948 ctggaggggtgtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 2008 gttcattctgcccttccaaa tcatctaagt gttatttaag gcactctgct gtttgtatga 2068 gatggttcatagaaattatg acaaagcctt tgttatccag gccatgggaa gag 2121 2 359 PRT Musmusculus 2 Met Arg Arg Pro Ser Leu Leu Ile Lys Asp Ile Cys Lys Cys ThrLeu 1 5 10 15 Val Ala Phe Gly Val Trp Leu Leu Tyr Ile Leu Ile Leu AsnTyr Thr 20 25 30 Ala Glu Glu Cys Asp Met Lys Arg Met His Tyr Val Asp ProAsp Arg 35 40 45 Ile Lys Arg Ala Gln Ser Tyr Ala Gln Glu Val Leu Gln LysGlu Cys 50 55 60 Arg Pro Arg Tyr Ala Lys Thr Ala Met Ala Leu Leu Phe GluAsp Arg 65 70 75 80 Tyr Ser Ile Asn Leu Glu Pro Phe Val Gln Lys Val ProThr Ala Ser 85 90 95 Glu Ala Glu Leu Lys Tyr Asp Pro Pro Phe Gly Phe ArgLys Phe Ser 100 105 110 Ser Lys Val Gln Ser Leu Leu Asp Met Leu Pro GluHis Asp Phe Pro 115 120 125 Glu His Leu Arg Ala Lys Ala Cys Lys Arg CysVal Val Val Gly Asn 130 135 140 Gly Gly Ile Leu His Gly Leu Glu Leu GlyHis Ala Leu Asn Gln Phe 145 150 155 160 Asp Val Val Ile Arg Leu Asn SerAla Pro Val Glu Gly Tyr Ser Glu 165 170 175 His Val Gly Asn Lys Thr ThrIle Arg Met Thr Tyr Pro Glu Gly Ala 180 185 190 Pro Leu Ser Asp Val GluTyr Tyr Ala Asn Asp Leu Phe Val Thr Val 195 200 205 Leu Phe Lys Ser ValAsp Phe Lys Trp Leu Gln Ala Met Val Lys Asn 210 215 220 Glu Ser Leu ProPhe Trp Val Arg Leu Phe Phe Trp Lys Gln Val Ala 225 230 235 240 Glu LysVal Pro Leu Gln Pro Lys His Phe Arg Ile Leu Asn Pro Val 245 250 255 IleIle Lys Glu Thr Ala Phe Asp Ile Leu Gln Tyr Ser Glu Pro Gln 260 265 270Ser Arg Phe Trp Gly His Asp Lys Asn Ile Pro Thr Ile Gly Val Ile 275 280285 Ala Val Val Leu Ala Thr His Leu Cys Asp Glu Val Ser Leu Ala Gly 290295 300 Phe Gly Tyr Asp Leu Ser Gln Pro Arg Thr Pro Leu His Tyr Phe Asp305 310 315 320 Ser Gln Cys Met Gly Ala Met His Trp Gln Val Met His AsnVal Thr 325 330 335 Thr Glu Thr Lys Phe Leu Leu Lys Leu Leu Lys Glu GlyVal Val Glu 340 345 350 Asp Leu Ser Gly Gly Ile His 355 3 17 DNAArtifical Sequence Synthetic DNA as 5′-primer 3 atgaaaagaa tgcacta 17 420 DNA Artificial Sequence Synthetic DNA as 3′-primer 4 tcagtggatgccgccgctga 20 5 18 PRT Mus musculus 5 Leu Leu Lys Leu Leu Lys Glu GlyVal Val Glu Asp Leu Ser Gly Gly 1 5 10 15 Ile His 6 48 PRT Mus musculus6 Cys Lys Arg Cys Val Val Val Gly Asn Gly Gly Ile Leu His Gly Leu 1 5 1015 Glu Leu Gly His Ala Leu Asn Gln Phe Asp Val Val Ile Arg Leu Asn 20 2530 Ser Ala Pro Val Glu Gly Tyr Ser Glu His Val Gly Asn Lys Thr Thr 35 4045 7 2359 DNA Homo sapiens CDS (278)..(1363) 7 ctgagcgggg gagcggcggcccccagctga atgggcgcga gagcggcgct gggggcgggt 60 gggggcgcgg ggtaccgggctggcggccgg ccggcgcccc ctcattagta tgcggacgaa 120 ggcggcgggc tgcgcggagcggcgtcccct gcagccgcgg accgaggcag cggcggcacc 180 tgccggccga gcaatgccaagtgagtacac ctatgtgaaa ctgagaagtg attgctcgag 240 gccttccctg caatggtacacccgagctca aagcaag atg aga agg ccc agc ttg 295 Met Arg Arg Pro Ser Leu 15 tta tta aaa gac atc ctc aaa tgt aca ttg ctt gtg ttt gga gtg tgg 343Leu Leu Lys Asp Ile Leu Lys Cys Thr Leu Leu Val Phe Gly Val Trp 10 15 20atc ctt tat atc ctc aag tta aat tat act act gaa gaa tgt gac atg 391 IleLeu Tyr Ile Leu Lys Leu Asn Tyr Thr Thr Glu Glu Cys Asp Met 25 30 35 aaaaaa atg cat tat gtg gac cct gac cgt gta aag aga gct cag aaa 439 Lys LysMet His Tyr Val Asp Pro Asp Arg Val Lys Arg Ala Gln Lys 40 45 50 tat gctcag caa gtc ttg cag aag gaa tgt cgt ccc aag ttt gcc aag 487 Tyr Ala GlnGln Val Leu Gln Lys Glu Cys Arg Pro Lys Phe Ala Lys 55 60 65 70 aca tcaatg gcg ctg tta ttt gag cac agg tat agc gtg gac tta ctc 535 Thr Ser MetAla Leu Leu Phe Glu His Arg Tyr Ser Val Asp Leu Leu 75 80 85 cct ttt gtgcag aag gcc ccc aaa gac agt gaa gct gag tcc aag tac 583 Pro Phe Val GlnLys Ala Pro Lys Asp Ser Glu Ala Glu Ser Lys Tyr 90 95 100 gat cct cctttt ggg ttc cgg aag ttc tcc agt aaa gtc cag acc ctc 631 Asp Pro Pro PheGly Phe Arg Lys Phe Ser Ser Lys Val Gln Thr Leu 105 110 115 ttg gaa ctcttg cca gag cac gac ctc cct gaa cac ttg aaa gcc aag 679 Leu Glu Leu LeuPro Glu His Asp Leu Pro Glu His Leu Lys Ala Lys 120 125 130 acc tgt cggcgc tgt gtg gtt att gga agc gga gga ata ctg cac gga 727 Thr Cys Arg ArgCys Val Val Ile Gly Ser Gly Gly Ile Leu His Gly 135 140 145 150 tta gaactg ggc cac acc ctg aac cag ttc gat gtt gtg ata agg tta 775 Leu Glu LeuGly His Thr Leu Asn Gln Phe Asp Val Val Ile Arg Leu 155 160 165 aac agtgca cca gtt gag gga tat tca gaa cat gtt gga aat aaa act 823 Asn Ser AlaPro Val Glu Gly Tyr Ser Glu His Val Gly Asn Lys Thr 170 175 180 act ataagg atg act tat cca gag ggc gca cca ctg tct gac ctt gaa 871 Thr Ile ArgMet Thr Tyr Pro Glu Gly Ala Pro Leu Ser Asp Leu Glu 185 190 195 tat tattcc aat gac tta ttt gtt gct gtt tta ttt aag agt gtt gat 919 Tyr Tyr SerAsn Asp Leu Phe Val Ala Val Leu Phe Lys Ser Val Asp 200 205 210 ttc aactgg ctt caa gca atg gta aaa aag gaa acc ctg cca ttc tgg 967 Phe Asn TrpLeu Gln Ala Met Val Lys Lys Glu Thr Leu Pro Phe Trp 215 220 225 230 gtacga ctc ttc ttt tgg aag cag gtg gca gaa aaa atc cca ctg cag 1015 Val ArgLeu Phe Phe Trp Lys Gln Val Ala Glu Lys Ile Pro Leu Gln 235 240 245 ccaaaa cat ttc agg att ttg aat cca gtt atc atc aaa gag act gcc 1063 Pro LysHis Phe Arg Ile Leu Asn Pro Val Ile Ile Lys Glu Thr Ala 250 255 260 tttgac atc ctt cag tac tca gag cct cag tca agg ttc tgg ggc cga 1111 Phe AspIle Leu Gln Tyr Ser Glu Pro Gln Ser Arg Phe Trp Gly Arg 265 270 275 gataag aac gtc ccc aca atc ggt gtc att gcc gtt gtc tta gcc aca 1159 Asp LysAsn Val Pro Thr Ile Gly Val Ile Ala Val Val Leu Ala Thr 280 285 290 catctg tgc gat gaa gtc agt ttg gcg ggt ttt gga tat gac ctc aat 1207 His LeuCys Asp Glu Val Ser Leu Ala Gly Phe Gly Tyr Asp Leu Asn 295 300 305 310caa ccc aga aca cct ttg cac tac ttc gac agt caa tgc atg gct gct 1255 GlnPro Arg Thr Pro Leu His Tyr Phe Asp Ser Gln Cys Met Ala Ala 315 320 325atg aac ttt cag acc atg cat aat gtg aca acg gaa acc aag ttc ctc 1303 MetAsn Phe Gln Thr Met His Asn Val Thr Thr Glu Thr Lys Phe Leu 330 335 340tta aag ctg gtc aaa gag gga gtg gtg aaa gat ctc agt gga ggc att 1351 LeuLys Leu Val Lys Glu Gly Val Val Lys Asp Leu Ser Gly Gly Ile 345 350 355gat cgt gaa ttt tgaacacaga aaacctcagt tgaaaatgca actctaactc 1403 Asp ArgGlu Phe 360 tgagagctgt ttttgacagc cttcttgatg tatttctcca tcctgcagatactttgaagt 1463 gcagctcatg tttttaactt ttaatttaaa aacacaaaaa aaattttagctcttcccact 1523 ttttttttcc tatttatttg aggtcagtgt ttgtttttgc acaccattttgtaaatgaaa 1583 cttaagaatt gaattggaaa gacttctcaa agagaattgt atgtaacgatgttgtattga 1643 tttttaagaa agtaatttaa tttgtaaaac ttctgctcgt ttacactgcacattgaatac 1703 aggtaactaa ttggaaggag aggggaggtc actcttttga tggtggccctgaacctcatt 1763 ctggttccct gctgcgctgc ttggtgtgac ccacggagga tccactcccaggatgacgtg 1823 ctccgtagct ctgctgctga tactgggtct gcgatgcagc ggcgtgaggcctgggctggt 1883 tggagaaggt cacaaccctt ctctgttggt ctgccttctg ctgaaagactcgagaaccaa 1943 ccagggaagc tgtcctggag gtccctggtc ggagagggac atagaatctgtgacctctga 2003 caactgtgaa gccaccctgg gctacagaaa ccacagtctt cccagcaattattacaattc 2063 ttgaattcct tggggatttt ttactgccct ttcaaagcac ttaagtgttagatctaacgt 2123 gttccagtgt ctgtctgagg tgacttaaaa aatcagaaca aaacttctattatccagagt 2183 catgggagag tacacccttt ccaggaataa tgttttggga aacactgaaatgaaatcttc 2243 ccagtattat aaattgtgta tttaaaaaaa agaaactttt ctgaatgcctacctggcggt 2303 gtataccagg cagtgtgcca gtttaaaaag atgaaaaaga ataaaaacttttgagg 2359 8 362 PRT Homo sapiens 8 Met Arg Arg Pro Ser Leu Leu Leu LysAsp Ile Leu Lys Cys Thr Leu 1 5 10 15 Leu Val Phe Gly Val Trp Ile LeuTyr Ile Leu Lys Leu Asn Tyr Thr 20 25 30 Thr Glu Glu Cys Asp Met Lys LysMet His Tyr Val Asp Pro Asp Arg 35 40 45 Val Lys Arg Ala Gln Lys Tyr AlaGln Gln Val Leu Gln Lys Glu Cys 50 55 60 Arg Pro Lys Phe Ala Lys Thr SerMet Ala Leu Leu Phe Glu His Arg 65 70 75 80 Tyr Ser Val Asp Leu Leu ProPhe Val Gln Lys Ala Pro Lys Asp Ser 85 90 95 Glu Ala Glu Ser Lys Tyr AspPro Pro Phe Gly Phe Arg Lys Phe Ser 100 105 110 Ser Lys Val Gln Thr LeuLeu Glu Leu Leu Pro Glu His Asp Leu Pro 115 120 125 Glu His Leu Lys AlaLys Thr Cys Arg Arg Cys Val Val Ile Gly Ser 130 135 140 Gly Gly Ile LeuHis Gly Leu Glu Leu Gly His Thr Leu Asn Gln Phe 145 150 155 160 Asp ValVal Ile Arg Leu Asn Ser Ala Pro Val Glu Gly Tyr Ser Glu 165 170 175 HisVal Gly Asn Lys Thr Thr Ile Arg Met Thr Tyr Pro Glu Gly Ala 180 185 190Pro Leu Ser Asp Leu Glu Tyr Tyr Ser Asn Asp Leu Phe Val Ala Val 195 200205 Leu Phe Lys Ser Val Asp Phe Asn Trp Leu Gln Ala Met Val Lys Lys 210215 220 Glu Thr Leu Pro Phe Trp Val Arg Leu Phe Phe Trp Lys Gln Val Ala225 230 235 240 Glu Lys Ile Pro Leu Gln Pro Lys His Phe Arg Ile Leu AsnPro Val 245 250 255 Ile Ile Lys Glu Thr Ala Phe Asp Ile Leu Gln Tyr SerGlu Pro Gln 260 265 270 Ser Arg Phe Trp Gly Arg Asp Lys Asn Val Pro ThrIle Gly Val Ile 275 280 285 Ala Val Val Leu Ala Thr His Leu Cys Asp GluVal Ser Leu Ala Gly 290 295 300 Phe Gly Tyr Asp Leu Asn Gln Pro Arg ThrPro Leu His Tyr Phe Asp 305 310 315 320 Ser Gln Cys Met Ala Ala Met AsnPhe Gln Thr Met His Asn Val Thr 325 330 335 Thr Glu Thr Lys Phe Leu LeuLys Leu Val Lys Glu Gly Val Val Lys 340 345 350 Asp Leu Ser Gly Gly IleAsp Arg Glu Phe 355 360 9 17 DNA Artificial Sequence Synthetic DNA 9atgaaaaaaa tgcatta 17 10 17 DNA Artificial Sequence Synthetic DNA 10tcaaaattca cgatcaa 17 11 48 PRT Homo sapiens 11 Cys Arg Arg Cys Val ValIle Gly Ser Gly Gly Ile Leu His Gly Leu 1 5 10 15 Glu Leu Gly His ThrLeu Asn Gln Phe Asp Val Val Ile Arg Leu Asn 20 25 30 Ser Ala Pro Val GlnGly Tyr Ser Glu His Val Gly Asn Lys Thr Thr 35 40 45 12 23 PRT Homosapiens 12 Pro Thr Ile Gly Val Ile Ala Val Val Leu Ala Thr His Leu CysAsp 1 5 10 15 Glu Val Ser Leu Ala Gly Phe 20

What is claimed is:
 1. An isolated sialyltransferase having thefollowing physico-chemical properties: (1) Activity: transfers sialicacid from a sialic acid donor selectively to a 3-hydroxyl group of agalactose residue contained in lactosylceramide as a sialic acidacceptor to produce ganglioside G_(M3); (2) Optimal reaction pH: 6.0 to7.0; and (3) Activation: the activity increases at least 1.5 times with10 mM of Mn²⁺ as compared with the case in the absence thereof.
 2. Anisolated sialyltransferase having a C-terminal amino acid sequence shownby SEQ ID NO: 5, and having an activity of transferring sialic acid to a3-hydroxyl group of a galactose residue.
 3. An isolatedsialyltransferase comprising an amino acid sequence shown by SEQ ID NO:6, and having an activity of transferring sialic acid to a 3-hydroxylgroup of a galactose residue.
 4. An isolated sialyltransferasecomprising an amino acid sequence shown by SEQ ID NO; 12, and having anactivity of transferring sialic acid to a 3-hydroxyl group of agalactose residue.
 5. The sialyltransferase according to claim 4,wherein said sialyltransferase is derived from human.
 6. Thesialyltransferase according to claim 1, wherein said sialic acid donoris cytidine 5′-monophosphate-sialic acid (CMP-sialic acid).
 7. Thesialyltransferase according to claim 1, wherein said sialyltransferaseis derived from human.
 8. An isolated sialyltransferase comprising apolypeptide having an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence shown by SEQ ID NO: 2, and (b)an amino acid sequence shown by SEQ ID NO: 2, which has thereinsubstitution, deletion, insertion or rearrangement of one or a few aminoacid residues, said sialyltransferase having an enzyme activity oftransferring sialic acid from a sialic acid donor selectively to a3-hydroxyl group of a galactose residue contained in lactosylceramide asa sialic acid acceptor to produce ganglioside G_(M3).
 9. An isolatedsialyltransferase comprising a polypeptide, wherein said polypeptide hasan amino acid sequence with not less than 65% homology with an aminoacid sequence shown by SEQ ID NO:
 8. 10. An isolated sialyltransferasehaving no transmembrane domain, said sialyltransferase having an enzymeactivity of transferring sialic acid from a sialic acid donorselectively to a 3-hydroxyl group of a galactose residue contained inlactosylceramide as a sialic acid acceptor to produce gangliosideG_(M3).
 11. The sialyltransferase according to claim 10, wherein saidsialyltransferase has an amino acid sequence comprising SEQ ID NO: 8with a transmembrane domain deletion.
 12. An isolated sialyltransferasehaving an amino acid sequence of amino acid numbers 38 to 359 in anamino acid sequence shown by SEQ ID NO:
 2. 13. An isolated DNA encodingall or a sialyltransferase-active part of the polypeptide of thesialyltransferase as defined in claim
 8. 14. An isolated DNA which has anucleotide sequence shown by SEQ ID NO;
 1. 15. An isolated DNA which hasa nucleotide sequence of base numbers 202 to 1278 in a nucleotidesequence shown by SEQ ID NO:
 1. 16. An isolated DNA encoding asialyltransferase-active homologous variant of an amino acid sequenceshown by SEQ ID NO:
 8. 17. An isolated DNA encoding asialyltransferase-active homologous variant of an amino acid sequence ofamino acid numbers 30 to 362 in the amino acid sequence shown by SEQ IDNO:
 8. 18. An isolated DNA which hybridizes with a polynucleotide havinga nucleotide sequence complementary to a nucleotide sequence shown bySEQ ID NO: 1 under stringent conditions.
 19. An isolated DNA whichhybridizes with a polynucleotide having a nucleotide sequencecomplementary to a nucleotide sequence of base numbers 202 to 1278 inthe nucleotide sequence shown by SEQ ID NO: 1 under stringentconditions.
 20. An isolated DNA which hybridizes with a polynucleotidecomplementary to the DNA as defined in claim 16 under stringentconditions.
 21. An isolated DNA which hybridizes with a polynucleotidecomplementary to the DNA as defined in claim 17 under stringentconditions.
 22. An isolated sialyltransferase comprising an amino acidsequence of amino acid numbers 41 to 362 in an amino acid sequence shownby SEQ ID NO: 8, and has an activity of transferring sialic acid to a3-hydroxyl group of a galactose residue.
 23. The sialyltransferaseaccording to claim 22, which comprises an amino acid sequence of aminoacid numbers 1 to 362 in amino acid sequence shown by SEQ ID NO:
 8. 24.The sialyltransferase according to claim 22, which has an activity oftransferring sialic acid to a 3-hydroxyl group of a galactose residuecontained in lactosylceramide to produce ganglioside G_(M3).
 25. Anisolated sialyltransferase comprising an amino acid sequence shown bySEQ ID NO:
 11. 26. An isolated polypeptide comprising an amino acidsequence of amino acid numbers 41 to 362 in amino acid sequence shown bySEQ ID NO:
 8. 27. An isolated polypeptide comprising an amino acidsequence of amino acid numbers 1 to 362 in amino acid sequence shown bySEQ ID NO:
 8. 28. An isolated DNA encoding the polypeptide as defined inclaim
 26. 29. An isolated DNA encoding the polypeptide as defined inclaim
 27. 30. An isolated DNA comprising a nucleotide sequence shown bySEQ ID NO:
 7. 31. An isolated DNA which hybridizes with a polynucleotidehaving a nucleotide sequence complementary to a nucleotide sequenceshown by SEQ ID NO; 7 under stringent conditions.
 32. An isolated DNAwhich hybridizes with a polynucleotide having a nucleotide sequencecomplementary to a nucleotide sequence of base numbers 275 to 1363 inthe nucleotide sequence shown by SEQ ID NO: 7 under stringentconditions.
 33. A recombinant vector comprising the DNA encoding thepolypeptide as defined in claim
 26. 34. A recombinant vector comprisingthe DNA encoding the polypeptide as defined in claim
 27. 35. Arecombinant vector comprising the DNA comprising a nucleotide sequenceshown by SEQ ID NO:
 7. 36. A recombinant vector comprising the DNAcomprising a nucleotide sequence shown by SEQ ID NO:
 1. 37. Atransformant into which a DNA is introduced, and in which the DNA can beexpressed, said DNA encoding the polypeptide as defined in claim
 26. 38.A transformant into which a DNA is introduced, and in which the DNA canbe expressed, said DNA encoding the polypeptide as defined in claim 27.39. A transformant into which a DNA is introduced, and in which the DNAcan be expressed, said DNA comprising a nucleotide sequence shown by SEQID NO;
 7. 40. A transformant into which a DNA is introduced, and inwhich the DNA can be expressed, said DNA comprising a nucleotidesequence shown by SEQ ID NO:
 1. 41. A method for producing asialyltransferase or a polypeptide thereof, comprising cultivating thetransformant as defined in claim 37 in a suitable medium, to produce andaccumulate in the culture the sialyltransferase or the polypeptidethereof encoded by the DNA, and collecting the sialyltransferase or thepolypeptide thereof from the culture.
 42. A method for producing asialyltransferase or a polypeptide thereof, comprising cultivating thetransformant as defined in claim 38 in a suitable medium, to produce andaccumulate in the culture the sialyltransferase or the polypeptidethereof encoded by the DNA, and collecting the sialyltransferase or thepolypeptide thereof from the culture.
 43. A method for producing asialyltransferase or a polypeptide thereof, comprising cultivating thetransformant as defined in claim 39 in a suitable medium, to produce andaccumulate in the culture the sialyltransferase or the polypeptidethereof encoded by the DNA, and collecting the sialyltransferase or thepolypeptide thereof from the culture.